Novel potent antimitotic heterocyclic ketones: Synthesis, antiproliferative activity, and structure-activity relationships

Article abstract of DOI:10.1016/j.bmcl.2007.04.048

We report the synthesis, antiproliferative activity, and SAR of novel heterocyclic ketones derived from carbazole sulfonamides. Most of the heterocyclic ketones showed strong cytotoxicities. (N-1-Methylindole-5-yl)-(3,4,5-trimethoxyphenyl)-methanone 8b gave the most potent cytotoxicity (9.2-26 nM) against seven human tumor cell lines. The mechanism of action of the heterocyclic ketones appears to involve targeting of tubulin, similar to that of CA-4 and different from the carbazole sulfonamides.

Full text of DOI:10.1016/j.bmcl.2007.04.048

Bioorganic & Medicinal Chemistry Letters 17 (2007) 3613–3617
Novel potent antimitotic heterocyclic ketones: Synthesis,
antiproliferative activity, and structure–activity relationships
Laixing Hu,a,b Jian-dong Jiang,b Jinrong Qu,c Yan Li,c Jie Jin,b Zhuo-rong Lib,*
and David W. Boykina,*
aDepartment of Chemistry, Georgia State University, Atlanta, GA 30303-3083, USA
bInstitute of Medicinal Biotechnology, Chinese Academy of Medical Sciences & Peking Union Medical College,
Beijing 100050, People’s Republic of China
cDepartment of Medicine, Mount Sinai School of Medicine, New York, NY 10029, USA
Received 23 March 2007; revised 15 April 2007; accepted 17 April 2007
Available online 25 April 2007
Abstract—We report the synthesis, antiproliferative activity, and SAR of novel heterocyclic ketones derived from carbazole sulfon-
amides. Most of the heterocyclic ketones showed strong cytotoxicities. (N-1-Methylindole-5-yl)-(3,4,5-trimethoxyphenyl)-metha-
none 8b gave the most potent cytotoxicity (9.2–26 nM) against seven human tumor cell lines. The mechanism of action of the
heterocyclic ketones appears to involve targeting of tubulin, similar to that of CA-4 and different from the carbazole sulfonamides.
Ó 2007 Elsevier Ltd. All rights reserved.
Antimitotic agents that target tubulin are effective and
widely used in treating cancer.1,2 Combretastatin A-4
(CA-4) (Chart 1) is a potent antimitotic agent, which
was isolated from the Africa Willow tree Combretum
caffrum.3 CA-4 strongly binds to the colchicine site of
tubulin and prevents tubulin polymerization.4 CA-4
inhibits cancer cell growth at low nanomolar concentra-
tions, including multi-drug-resistant cancer cell lines. A
water soluble prodrug of CA-4 has shown promising
results in phase I human cancer clinical trials.5,6 The rel-
atively simple structure of CA-4 as well as its selective
antivascular activity have stimulated significant research
efforts focused on the identification of new CA-4 ana-
logues that exhibit more potent activities and better
pharmacological profiles.7,8 Recently, another two CA-
4 analogues, CA-1P and AVE-8062 (Chart 1), have
entered clinical trials.9
cytotoxicity.11,12 CA-4 is prone to isomerize to the E-iso-
mer (trans) during storage and administration, which
dramatically reduces its antiproliferative activities.13
By replacement of the ethene bridge of CA-4 with a car-
bonyl group, Phenstatin and Hydroxyphenstatin (two
benzophenone analogues) (Chart 1) have been found
to exhibit potent anticancer and comparable antitubulin
activities with CA-4.14,15 The sp2-hybridized carbonyl
group in Phenstatin and Hydroxyphenstatin molecules
not only constrains the quasi ‘cis’ orientation, but often
improves chemical stability and water solubility.15b 20-
or 30-Aminobenzophenone analogues (Chart 1) also
showed significant cytotoxicity against many human
cancer cell lines.16,17 More interestingly, 2-aroyl-5-meth-
oxyindoles and 3-aroyl-6-methoxyindoles (Chart 1) have
shown inhibition of tubulin polymerization, potential
antivascular activity, and oral effectiveness in in vivo
tumor models.18,19
CA-4 as an antimitotic agent displays poor in vivo anti-
cancer efficacy, partly due to its high lipophilicity and
low aqueous solubility.10 SAR studies have shown that
the Z (cis) orientation of the olefin is essential for potent
We recently reported a series of carbazole sulfonamide
antimitotic agents structurally related to CA-4.20,21
The N-9-ethyl-3,4,5-trimethoxyphenyl-carbazolesulfon-
amide analogues 1–3 and the 2,6-dimethoxypyridin-3-yl
analogue 4 (Chart 1) displayed potent antiproliferative
activities against several human cancer cell lines. The
lead compound 1 also showed significant anticancer
activity in two in vivo models. However, the lead com-
pound 1 does not inhibit tubulin polymerization, which
Keywords: Antiproliferative; Solid tumors; Tubulin; Heterocyclic
ketones.
*
Corresponding authors. Tel.: +1 404 651 3798, fax: +1 404 651 1416
(D.W.B.); tel.: +86 10 6302 7185; fax: +86 10 6301 7302 (Z.-R.L.);
e-mail addresses: l-z-r@263.net; dboykin@gsu.edu
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.04.048

3614
L. Hu et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3613–3617
Chart 1. Antimitotic agents: CA-4, CA-4 analogues and carbazole sulfonamides.
suggests the mode of action is different from CA-4.20
For the purpose of further study of SAR of this series
of carbazole sulfonamides and discovery of more effec-
tive antimitotic agents, we designed a series of aroyl car-
bazoles through replacing the sulfonamide linkage of
carbazole sulfonamides with the carbonyl group. Also,
we include aroyl indoles, benzofuran, and benzothioph-
ene analogues to further evaluate the SAR in this series.
Here, we report the synthesis and cytotoxic activities
against human cancer cells of this series of novel hetero-
cyclic ketone compounds.
The synthesized heterocyclic ketones (Table 1) were
evaluated for their cytotoxicities against the CEM
leukemia cell line.20 We first evaluated the effect of
replacement of the sulfonamide group with the car-
bonyl group in the carbazole sulfonamide lead com-
pound 1 on cytotoxicity. Newly synthesized ketone
8a exhibited a slight loss of potency as compared
to lead compound 1, IC50 value of 90 vs 56 nM.20
The corresponding carbinol 7a also showed compara-
ble activity to lead compound 1. These results suggest
that the carbonyl group is tolerated as a replacement
of the sulfonamide group with retention of potent
cytotoxic activity. Then, replacement of the 9-ethylc-
arbazole ring of ketone 8a with N-methylindole ring
yielded the 5-aroyl-N-1-methylindole analogue 8b. It
is gratifying to note that compound 8b gave signifi-
cantly increased potency (5-fold) as compared to 8a
and also a 3-fold increase of activity compared to
lead compound 1. A similar tendency has also been
shown for the N-1-methylindole methanol 7b, which
displayed an IC50 value of 59 nM, almost the same
value as lead compound 1. When the position of
the carbonyl group is changed to the C-6 position
on the indole ring substantial potency is maintained.
However, changing the keto group to the C-7 posi-
tion of the indole ring (8d) was not tolerated and de-
creased cytotoxicity by 17 times as compared to 8b
was noted, suggesting that the effect of the ortho-ste-
ric hindrance is likely critical for cytotoxicity.
The general method for the synthesis of heterocyclic
ketone compounds is shown in Schemes 1 and 2.16
The Grignard reaction of various substituted phenyl or
2,6-dimethoxypyridinyl magnesium bromide with
N-9-ethyl-3-carbazolecarboxaldehyde, N-1-methyl-5, or
6, or 7-indolecarboxaldehyde, 5-benzofurancarboxalde-
hyde, and 5-benzothiophenecarboxaldehyde yielded the
corresponding diaromatic methanols 7a–k, 10a and
10b. We obtained the corresponding desired ketones
8a–k, 11a and 11b by pyridinium dichromate (PDC)
oxidation of the carbinols. For the synthesis of (N-1H-
indole-5-yl)-(3, 4, 5-trimethoxyphenyl)-methanone 17,
it was necessary to first protect the NH group of N-
1H-5-indolecarboxaldehyde 13 with TBDMS. Then,
following the general procedure of Grignard reaction,
oxidation, and deprotection with Bu4NF, the ketone
17 was obtained (Scheme 3).

L. Hu et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3613–3617
3615
Scheme 1. Reagents and conditions: (a) THF, 0 °C; (b) PDC, CH2Cl2, 25 °C.
Scheme 2. Reagents and conditions: (a) THF, 0 °C; (b) PDC, CH2Cl2, 25 °C.
Scheme 3. Reagents and conditions: (a) THF, 0 °C; (b) PDC, CH2Cl2, 25 °C; (c) TBDMSCl, t-BuOK, THF, 0–25 °C; (d) Bu4NF, THF.
The influence of various substitutions on the phenyl ring
was also examined. 4-Methoxy, 3,5-, 3,4-,and 2,5-dime-
thoxy substitutions on the phenyl ring (8e, 8f, 8g and 8i)
led to significant loss of potencies more than a 50-fold
reduction in activity in comparison with the 3,4,5-tri-
methoxyphenyl compound 8b. However, 2,4-dimethoxy
substitutions of phenyl ring (8h) gave an IC50 value of
102 nM, only a 5-fold loss of activity as compared to
8b. The SAR of this series of compounds is different
from that of the carbazole sulfonamides. For example,
we reported that the 2,4- and 2,5-dimethoxy phenyl
substituted carbazole sulfonamides 2 and 3 showed sim-
ilar cytotoxicities to that of the 3,4,5-trimethoxyphenyl
lead compound 1.20 The SAR of the ketone series of
compounds is consistent with that of CA-4 analogues,
where it is well known that the 3,4,5-trimethoxy substit-
uents of A ring of CA-4 are indispensable for potent
cytotoxicity.11 From these observations, the mechanism
of action of this series of compounds appears to be dif-
ferent from the carbazole sulfonamides and similar to
that of CA-4 analogues.
than a 10-fold decrease in potency giving IC50 values
from 225 to 303 nM, compared to the IC50 value of
18 nM for 8b. Similar results were also noted in the car-
bazole sulfonamide series.20
In the 2,6-dimethoxypyridinyl series, ketone compound
11a led to 4.5-fold loss of potency in comparison with
the corresponding carbazole sulfonamide 4.21 However,
it is much better than the ethene compound
(IC50 > 2 lM). In contrast, the (2,6-dimethoxypyridin-
3-yl)-N-1-methylindole-ketone 11b has decreased cyto-
toxicity as compared to the corresponding carbazole
ketone 11a, the IC50 value of 1012 versus 555 nM, indi-
cating that it is different from 3,4,5-trimethoxyphenyl
substitution. It is noteworthy that compound 11b is
less potent by 10-fold than the 2,4-dimethoxyphenyl
indole compound 8h. These results suggest that the
2,6-dimethoxypyridinyl is not suitable for ketone and
ethene compounds to achieve potent cytotoxicity, per-
haps due to ortho-steric hindrance and meta-nitrogen
electron-withdrawing effects. The corresponding indole
sulfonamide also showed much decreased activity.21
Only 2,6-dimethoxypyridinyl carbazole sulfonamide 4
has comparable activity with the lead compound 1,
and it is likely that the mode of action of 4 is different
from lead compound 1.
In order to understand the effect of the nitrogen of the
indole ring, we synthesized the N-1H-indole, benzofu-
ran, and benzothiophene analogues 17, 8j, and 8k. It
was not surprising that these analogues resulted in more

3616
L. Hu et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3613–3617
Table 1. Antiproliferative activity of new compounds in CEM Leukemia cells
a
Cytotoxicity IC50 (nM)
Compound
Aryl type
X
R
Y
Z
1
I
C
C
C
N
C
C
C
C
C
C
C
C
C
C
C
C
C
C
N
N
3,4,5-OMe3
2,4-OMe2
2,5-OMe2
2,6-OMe2
3,4,5-OMe3
3,4,5-OMe3
3,4,5-OMe3
3,4,5-OMe3
3,4,5-OMe3
3,4,5-OMe3
3,5-OMe2
3,4-OMe2
2,5-OMe2
2,4-OMe2
4-OMe
SO2NH
SO2NH
SO2NH
SO2NH
CHOH
CO
/
56
57
2
I
/
3
I
/
61
4
7a
I
/
122
192
90
I
/
8a
7b
I
/
II
II
II
II
II
II
II
II
II
II
II
II
I
50-CHOH
50-CO
60-CO
70-CO
50-CO
50-CO
50-CO
50-CO
50-CO
50-CO
50-CO
50-CO
CO
NMe
NMe
NMe
NMe
NMe
NMe
NMe
NMe
NMe
NH
O
59
18
8b
8c
30
8d
8e
307
1693
1693
2370
102
1131
225
288
303
555
1012
7.2
8f
8g
8h
8i
17
3,4,5-OMe3
3,4,5-OMe3
3,4,5-OMe3
2,6-OMe2
2,6-OMe2
8j
8k
S
11a
11b
/
NMe
II
50-CO
Podophyllotoxin
CA-4
1.9
a Values were determined as described in Ref. 20.
Several analogues 8a, 7b, 8b, 8c, and 8h exhibit strong
antiproliferative activity against CEM leukemia cells;
consequently, we further evaluated their activity against
a panel of several other human cancer cell lines in vitro.
Table 2 contains these results along with comparative
data for lead compound 1, CA-4, and podophyllo-
toxin.20 Although carbazole ketone 8a is slightly less
potent than lead compound 1 against CEM and Molt-
3 leukemia cell lines, it shows 2- to 15-fold stronger
activity than 1 against the five human solid tumor cell
lines. The IC50 values of the most potent indole ketone
compound 8b were between 9.2 and 26 nM against the
panel of seven cell lines studied. Interestingly, com-
pound 8b is slightly more active against MCF-7 breast
cancer cell line in comparison to CA-4 and podophyllo-
toxin. Compound 8b is two times more potent than
podophyllotoxin against DU-145 prostate cancer cell
line and showed comparable activity in other five cell
lines with that of podophyllotoxin. Compound 8b also
is 6–23 times more active than lead compound 1 against
the five human solid tumor cell lines. The carbinol 7b
and 6-indole ketone 8c also showed more activity than
lead compound 1 in inhibiting solid tumor cell lines.
The 2,4-dimethoxy substituted indole compound 8h is
more potent than lead compound 1 against Bel-7402
hepatoma and DU-145 prostate cancer cells and showed
comparable activity in other cell lines to that of 1. These
results show that heterocyclic ketones are more effective
against solid tumor cell lines than lead compound 1. The
sensitivity of this series of compounds is similar to that
of CA-4 and Podophyllotoxin.
We have noted that the SAR of this series of heterocyclic
ketones is different from that of the carbazole sulfon-
Table 2. Antiproliferative activities of 8a, 7b, 8b, 8c, 8h, 1, CA-4, and Podophyllotoxin in human tumor cell lines
Cell linea
b
IC50 (nM)
8a
90
7b
59
8b
8c
8h
1
CA-4
Podophyllotoxin
CEM
18
18
21
30
18
27
24
75
75
27
102
24
56
20
1.9
9.3
10
7.2
14
Molt-3
Bel-7402
MCF-7
DU-145
PC-3
51
90
38
88
44
85
201
89
9.1
15
26
39
44
9.2
12
103
117
59
26
25
15
34
603
201
89
9.3
2.8
2.5
52
10
103
21
203
118
DND-1
12
a CEM; Molt-3, T-cell leukemia; Bel-7402, hepatoma; MCF-7, breast cancer; DU-145, PC-3, prostate cancer; DND-1, melanoma.
b Values were determined as described in Ref. 20.

L. Hu et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3613–3617
3617
0.35
0.3
Biotechnology and Drug Design for support of this
work.
0.25
0.2
Vin(20uM)
PTX(20uM)
Control
References and notes
8a(5uM)
8a(4uM)
8a(3uM)
8a(2uM)
8a(1uM)
0.15
0.1
1. Jordan, M. A.; Wilson, L. Nat. Rev. Cancer 2004, 4, 253.
2. Beckers, T.; Mahboobi, S. Drugs of the future 2003, 28,
767.
3. (a) Pettit, G. R.; Cragg, G. M.; Singh, S. B. J. Nat. Prod.
1987, 50, 386; (b) Pettit, G. R.; Singh, S. B.; Hamel, E.;
Lin, C. M.; Alberts, D. S.; Garcia-Kendall, D. Experientia
1989, 45, 209.
4. Lin, C. M.; Ho, H. H.; Pettit, G. R.; Hamel, E.
Biochemistry 1989, 28, 6984.
5. Pettit, G. R.; Temple, C., Jr.; Narayanan, V. L.; Varma,
R.; Boyd, M. R.; Rener, G. A.; Bansal, N. Anti-Cancer
Drug Des. 1995, 10, 299.
6. Young, S. L.; Chaplin, D. J. Expert Opin. Invest. Drugs
2004, 13, 1171.
0.05
0
0
2
4
6
8
10 12 14 16 18 20 22 24 26 28 30
Time (min)
Figure 1. Effect of 8a on tubulin assembly.20 Free purified b-tubulin in
reaction buffer was incubated with GTP and Mg2+ for assembly in the
absence or presence of 8a (1–5 lM), vincristine (20 lM) or paclitaxel
(20 lM). Tubulin assembly was determined every 2 min by O.D. at
340 nm. Each point represents the mean of two independent
experiments.
7. Tron, G. C.; Pirali, T.; Sorba, G.; Pagliai, F.; Busacca, S.;
Genazzani, A. A. J. Med. Chem. 2006, 49, 3033.
8. (a) Nam, N. H. Curr. Med. Chem. 2003, 10, 1697; (b)
Hsieh, H. P.; Liou, J. P.; Mahindroo, N. Curr. Pharm.
Des. 2005, 11, 1655.
9. Thorpe, P. E.; Chaplin, D. J.; Blakey, D. C. Cancer Res.
2003, 63, 1144.
10. Tozer, G. M.; Kanthou, C.; Parkins, C. S.; Hill, S. A. Int.
J. Exp. Pathol. 2002, 83, 21.
amides and similar to CA-4 analogues. The mechanism of
actionofthisseriesofketonesislikelysimilartoCA-4ana-
logues by targeting on tubulin. To investigate whether the
antiproliferative activities of these compounds were
related to interaction with tubulin, carbazole ketone com-
pound 8a was evaluated for inhibition of polymerization
of purified tubulin in a cell-free system. The results are
shown in Figure 1. Vincristine was used as a positive con-
trol. Vincristine inhibited tubulin polymerization by 87%
at 20 lM. The effect of 8a on tubulin assembly was exam-
ined at concentrations from 1 to 5 lM. The results showed
that compound 8a inhibited tubulin polymerization in a
dose-dependent manner. The IC50 value of inhibition of
tubulin polymerization of compound 8a was 3.4 lM; sim-
ilar to that of CA-4 at 1.2 lM.14 It is significantly lower
than that of the lead compound 1 which only weakly af-
fected tubulin polymerization, even at very high
concentration.20
11. Cushman, M.; Nagarathnam, D.; Gopal, D.; Chakraborti,
A. K.; Lin, C. M.; Hamel, E. J. Med. Chem. 1991, 34,
2579.
12. Cushman, M.; Nagarathnam, D.; Gopal, D.; He, H.-M.;
Lin, C. M.; Hamel, E. J. Med. Chem. 1992, 35, 2293.
13. Pettit, G. R.; Rhodes, M. R.; Herald, D. L.; Chaplin, D.
J.; Stratford, M. R.; Hamel, E.; Pettit, R. K.; Chapuis, J.
C.; Oliva, D. Anti-cancer Drug Des. 1998, 13, 981.
14. Pettit, G. R.; Toki, B.; Herald, D. L.; Verdier-Pinard, P.;
Boyd, M. R.; Hamel, E.; Pettit, R. K. J. Med. Chem. 1998,
41, 1688.
15. (a) Pettit, G. R.; Grealish, M. P.; Herald, D. L.; Boyd, M.
R.; Hamel, E.; Pettit, R. K. J. Med. Chem. 2000, 43, 2731;
(b) Pettit, G. R.; Lippert, J. W.; Herald, D. L. J. Org.
Chem. 2000, 65, 7438.
In summary, a series of novel heterocyclic ketones were
synthesized by replacing the sulfonamide linkage with a
carbonyl group in the lead carbazole sulfonamide 1.
Most of the heterocyclic ketones showed strong antipro-
liferative activities against CEM leukemia cells. Several
compounds were more effective than lead compound 1
against five solid tumor cell lines. The most potent com-
pound 8b displayed comparable antiproliferative activi-
ties with that of CA-4 and podophyllotoxin. The SAR of
the heterocyclic ketones is different from the carbazole
sulfonamides and similar to that of CA-4 analogues.
The carbazole ketone compound 8a strongly inhibits
tubulin assembly. Further studies of the mechanism of
action of this series of novel heterocyclic ketones and
evaluation of 8a and 8b in vivo are underway.
16. (a) Liou, J. P.; Chang, C. W.; Song, J. S.; Yang, Y. N.;
Yeh, C. F.; Tseng, H. Y.; Lo, Y. K.; Chang, Y. L.; Chang,
C. M.; Hsieh, H. P. J. Med. Chem. 2002, 45, 2556; (b)
Hsieh, H. P.; Liou, J. P.; Lin, Y. T.; Mahindroo, N.;
Chang, J. Y.; Yang, Y. N.; Chern, S. S.; Tan, U. K.;
Chang, C. W.; Chen, T. W.; Lin, C. H.; Chang, Y. Y.;
Wang, C. C. Bioorg. Med. Chem. Lett. 2003, 13, 101.
17. Liou, J. P.; Chang, J. Y.; Chang, C. W.; Chang, C. Y.;
Mahindroo, N.; Kuo, F. M.; Hsieh, H. P. J. Med. Chem.
2004, 47, 2897.
18. Mahboobi, S.; Pongratz, H.; Hufsky, H.; Hockemeyer, J.;
Frieser, M.; Lyssenko, A.; Paper, D. H.; Burgermeister, J.;
¨
Bo¨hmer, F. D.; Fiebig, H. H.; Burger, A. M.; Baasner, S.;
Beckers, T. J. Med. Chem. 2001, 44, 4535.
19. (a) Liou, J. P.; Chang, Y. L.; Kuo, F. M.; Chang, C. W.;
Tseng, H. Y.; Wang, C. C.; Yang, Y. N.; Chang, J. Y.;
Lee, S. J.; Hsieh, H. P. J. Med. Chem. 2004, 47, 4247; (b)
Dupeyre, G.; Chabot, G. G.; Thoret, S.; Cachet, X.;
Seguin, J.; Guenard, D.; Tillequin, F.; Scherman, D.;
Koch, M.; Michel, S. Bioorg. Med. Chem. 2006, 14, 4410.
20. Hu, L.; Li, Z. R.; Li, Y.; Qu, J.; Ling, Y. H.; Jiang, J. D.;
Boykin, D. W. J. Med. Chem. 2006, 49, 6273.
Acknowledgments
We thank the National Natural Science Foundation of
the PR China (30500630) and the Technology Develop-
ment Program of the Georgia State University Center of
21. Hu, L.; Li, Z. R.; Wang, Y. M.; Wu, Y.; Jiang, J. D.;
Boykin, D. W. Bioorg. Med. Chem. Lett. 2007, 17, 1193.